This invention relates to a microorganisms lactamase enzyme obtained therefrom, and their use.
The bicyclic xcex3-lactam, 2-azabicyclo 2.2.1hept-5-en-3-one, is a useful synthon hat can be used for the production of carbocyclic nucleosides which are gaining in importance as therapeutic agents. Published areas to which such nucleosides are being targeted include antivirals (e.g. Vince and Hua. J. Med. Chain., 33:17-21 (1990), againts e.g. HIV) and cardiac vasodilators (adenosine agonists). A major benefit of the carbocyclyc in such agents is its resistance to breakdown by enzymes in the body. By comparison, naturally-occurring ribosyl nucleosides may be more readily cleaved by nucleases, so that their bioactivity is lost.
Although carbocyclic nucleosides are known in nature e.g. Aristeromycin from Streptomyces citricolor, natural yields tend to be low and the isolated products have then to be further manipulated to obtain more useful compounds. A more econonic route is to synthesise the required compounds chemically, sarting from the xcex3-lactam. However, as chemically synthesised, xcex3-lactam is racemic By conventional synthesis, the ultimate drug will also be a mixture of enantiomers, which causes regulatory concerns if one of the enantiomers is not very active or causes unwanted side-efects. There is a need therefore to put a step into the synthesis where either of the two enantiomers of a racemic synthon can be isolated and the rest of the drug then built on it.
An effective way of doing this is to use an enzyme to selectively hydrolyse one enantiomer of the racemic xcex3-lactam across the amide bond, to give the cyclic amino acid compound and leave the other enantiomer. The remaining lactam can then be readily separated from the amino acid product by extraction into dichloromethane, purfied by crystailltisation and used in subsequent downstream chemistry to build up the required drug. By careful selection of the right enzyme it is possible to find an enzyme highly selectively for only one of the lactam enantiomers such that at marginally greater than 50% conversion, lactam of high ee ( greater than 90%) remains. Enzymes have been found that are selective for either of the two enantiomers.
EP-A-0424064 discloses methods for carrying out the above described resolution and provides two organisms that produce enzymes that have the different selectivities. A Rhodococcus strain produces an enzyme which hydrolyses the (xe2x88x92) lactam, enabling the (+) lactam to be isolated for further use. whereas a Pseudomonad produces an emzyme which hydrolyses the (+) lactam, enabling isolation of the (xe2x88x92) lactam.
Further enzymes that carry out these selective hydrolyses have also been described in the literature. Thus Taylor et al, Tetrahedron: Asymmetry, 4 (6):1117-1128 (1993), describe an enzyme selective for hydrolysis of the (+) lactam from a strain of Pseudonomas fluorescens and an enzyme selective for the (xe2x88x92) lactam from a strain of Aureobacterium. A further enzyme selective for the hydrolysis of the (+) lactam has been described by Brabban et al. J. Ind. Microbiology. 16:8-14 (1996).
In order to develop a robust industrial biotransformation process, it is desirable to use an enzyme or whole cell biocatalyst that is relatively stable. This can enable biocatalyst recycling and re-use through immobilisation, thus greatly reducing biocatalyst cost and enabling handling of the biocatalyst on a large scale without significant losses of activity. It is also often found that more stable biocatalysts are better able to tolerate high substrate and/or product concentrations without inactivation. This then enables biotransformation to be run at the highest concentration of reactants possible, given kinetic and handling constraints. This has two advantages: it results in minimal reactor volume requirements and also minimises liquid handing volumes during product work-up.
Taylor et al, supra. describe a lactamase from Aireobacterium especies that is very stable at elevated temperatures and which selectively hydrolyses the (xe2x88x92) xcex3-lactam, giving the (+) xcex3-lactam and (xe2x88x92) amino acid as a product. The enzyme from this organism has been immobilised and maintains its stability over months of operation. No enzyme with good stability and the opposite selectivity is known, although Brabban at al, Supra, screened a number of different potential isolates. Previous work with Pseudomonad type organisms displaying the required lactamase activity had shown them to have poor stability. This is unfortunate since it is the (xe2x88x92) xcex3-lactam which is the more usefwl synthon, having the more natural stereochemistry and making it easier to build up functionality than for instance the (xe2x88x92) amino acid formed by the action of the Aireobacterium lactamase. There is therefore a need for a stable xcex3-lactamase with high selectivity for the hydrolysis of the (+) bicyclic xcex3-lactam.
Surprisingly, it has been found that a strain of Comamonas acidivorans, which was isolated from the environment, produces an enzyme of high potential for use in an industrial process for resolution of the required xcex3-lactam. This enzyme is not only much more temperature-stable than previously identified (+) xcex3-lactamase, but it also enables the bioresolution to be carried out at very high substrate/product concentrations. This organism has been deposited at the NCIMB, 23 St. Machar Street, Aberdeen, UK, on 30th August 1996, under the terms of the Budapest Treaty, where it has been given the accession number NCIMB 40827.
The gene encoding the xcex3-lactamase has been isolated and sequenced (see SEQ ID NO:1), and the enzyme""s amino-acid sequence derived (see SEQ ID NO:2). This invention relates to compounds having this structure, and fragments thereof having the same activity, as will be readily evident to one of ordinary skill in the art. The novel enzyme is characterised by its stability, i.e. one or more of the following:
greater than 85% retention of activity after being held at 40xc2x0 C. for 4 hours or greater than 30% activity after being held at 60xc2x0 C. for 4 hours;
hydrolysis at an initial concentration of 100 g racemic lactam plus 300 ml buffer and continuing to at least 90% hydrolysis of the (+) lactam with less than 5% hydrolysis of the (xe2x88x92) lactam.